The medicinal activity of lyophilized aqueous seed extract of Lepidium sativum L. in an androgenic alopecia model

This study evaluated the topical effect of Lepidium sativum lyophilized seed extract (LSLE) towards Sustanon-induced alopecia in male adult Wistar albino rats in vivo, compared to minoxidil topical reference standard drug (MRD). LC–MS/MS together with molecular networking was used to profile the metabolites of LSLE. LSLE treated group revealed significant changes in alopecia related biomarkers, perturbation of androgenic markers; decline in testosterone level and elevation in 5α-reductase (5-AR); decline in the cholesterol level. On the other hand, LSLE treated group showed improvement in vascular markers; CTGF, FGF and VEGF. Groups treated topically with minoxidil and LSLE showed significant improvement in hair length. LC–MS/MS profile of LSLE tentatively identified 17 constituents: mainly glucosinolates, flavonoid glycosides, alkaloids and phenolic acids. The results point to the potential role of LSLE in the treatment of alopecia through decreasing 5(alpha)-dihydrotestosterone levels. Molecular docking was attempted to evaluate the probable binding mode of identified compounds to androgen receptor (PDB code: 4K7A).

Alopecia is a dermatological illness that has been known for over a thousand years and it is a common concern in both cosmetic and basic health care. Androgenic alopecia (AGA) is considered the most prevalent type of hair loss and is commonly used to define the condition of scalp hair loss in both males and females who are genetically susceptible to it 1 . It is characterized by the increased activity of the 5α-reductase (5AR) enzyme which accelerates the reduction of testosterone to 5α-dihydrotestosterone (DHT). Despite the fact that androgens are responsible for some secondary sexual characteristics such as facial hair growth, DHT miniaturizes androgensensitive follicles in the scalp causing thinning of the scalp hair 2 . Meanwhile, oxidative stress was found to accelerate cell senescence of dermal papilla cells via stimulating of the apoptotic pathways within hair follicles, resulting in faster hair loss in AGA. Thus, antioxidants could be regarded as a beneficial auxiliary treatment to hinder hair loss speed in AGA 3 .
Finasteride and minoxidil are both FDA-approved to treat AGA. Finasteride inhibits the 5α-reductase activity while minoxidil can enhance hair growth via vasodilatation caused by opening potassium channels located on smooth muscle cells of peripheral arteries with gradual slowing of circulation. Minoxidil topical solution is a clinically approved and effective hair growth stimulant. It can also reduce hair loss and maintain hair growth 4 .
Several Brassicaceae plants have been reported to promote hair growth in AGA treatment such as Brassica oleracea extract, its glucosinolate rich fraction 5 , wasabi derived 6-methylsulfinylhexyl isothiocyanate 6 and watercress extract 7 . Meanwhile, Lepidium sativum also known as garden cress exhibited antiandrogenic activity 8 . Animals. Male adult Wistar albino rats weighing on average 200-250 g were purchased from the Egyptian Organization for Biological Products and Vaccines Egypt. The animals were caged in plexiglass cages at a temperature of 25 ± 2 °C. They were exposed to a repeated cycle of 12 h of illumination followed by 12 h of darkness. They were continuously provided with water, as well as a standard pellet diet. All the performed procedures were in alignment with the guide for the care and use of laboratory animals at the US National Institute of Health (NIH Publication No. 85-23, revised 2011). The whole experimental work was approved by the Ethics Committee for Animal Experimentation at the Faculty of Pharmacy, MSA University (PH1/EC1/2022PD) and was conducted in accordance with the ARRIVE guidelines.
Drugs. Sustanon 250 (from Organon) and 5% minoxidil were purchased from the from a retail pharmacy and the production date checked before use.

Induction of alopecia.
At the start of the experiment part of the dorsal hair was clipped and then carefully shaved. Alopecia was then induced by subcutaneous (S.C.) injection of Sustanon dissolved in corn oil at a dose of 1 mg/kg on daily basis for a period of 21 days. The selected dose together with the utilized route of administration were adopted from literature 13,14 . Experimental design. Forty-two male Wistar albino rats were randomly placed into four distinct groups.
The rats were anesthetized then selected portions of their skin were clipped and carefully shaved with razors. Hair growth rates were observed daily. Group I (n = 7) served as a control group; rats had part of their dorsal hair clipped, shaved then received corn oil vehicle S.C. for 21 days during which their shaved portion of the skin was sprayed with saline (0.9% NaCl). The rest of the study groups had alopecia induced. Group II (n = 7) rats were sprayed with saline concurrently with Sustanon for 21 days while Group III (n = 14) had 5% minoxidil (2 sprays/ rat as that was the recommended dose from the manufacturer) and Group IV (n = 14) had LSLE (4% in saline Biochemical assays. The blood samples were used for the measurement of cholesterol levels. A commercially available kit was used for this measurement (Biovision, USA; #K4436-100). The kit employed the sandwich ELISA principle, and the procedures were done in accordance with the manufacturer's instructions. Cholesterol levels were finally estimated as ng/ml.
The tissue homogenate was used for the assessment of 5AR and the growth factors levels. 5AR and CTGF were assayed using sandwich ELISA from commercially available kits (Lifespan Biosciences, USA; #LS-F6847; Novus Biologicals, USA; #NBP2-75011, respectively). Both were used according to the manufacturer's guidelines and were sensitive to the pg/ml range.
The tissue levels of fibroblast growth factor (FGF) were determined by gene expression analysis via SuperScript IV One-Step RT-PCR kit (Thermo Fisher Scientific, USA, #12594100). The forward primer was 5' ATC CTG CCG ACT CCG CTC TA3' and the reverse was 5'CCT TTT GAT TTA AGG CCA CGA ACA 3' . The 2 -ΔΔCt approach was used to compare the CT of each sample with that of the control group.
The Western blotting technique was used to detect the tissue levels of vascular endothelial growth factor (VEGF). After sonicating the homogenate on ice to ensure cell lysis, it was centrifuged at 4 °C for 20 min at 15,000 rpm. Protein was isolated using the Ready Prep protein extraction kit (Bio-Rad, USA; #163-2086). The total protein content was determined using the Bradford assay, and 20 μg of protein was separated on SDS-PAGE (10% polyacrylamide gel) and transferred to polyvinylidene difluoride membranes (Pierce, Rockford, IL, USA) using a Bio-Rad Trans-Blot system. The membrane was blocked for 1 h with a blocking solution (20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.1% Tween 20, and 4% bovine serum albumin). After that, the membrane was washed twice with a wash buffer (20 mM Tris-Cl (pH 7.5) and 150 mM NaCl). The membrane was then incubated overnight at 4 °C with either the VEGF primary antibody (Abcam, UK, #ab46154) or β-actin primary antibody (Thermo Fisher Scientific Inc.) (Rockford, IL, USA). The membrane was then cleaned using wash steps. Following that, secondary antibodies labelled with horse radish peroxidase were added. The membranes were left at 25 °C for 1 h before being treated with luminol. The ChemiDoc imaging system with Image Lab software version 5.1 was used to measure the band intensity (Bio-Rad Laboratories Inc., Hercules, CA, USA). After normalization to β-actin protein levels, the results are displayed as arbitrary units.
Histopathological assessment of hair follicles. A 4 mm punch biopsy was used for sectioning samples which were fixed in formalin 10% for 24 h, dehydrated with increasing concentrations of alcohol, then sectioned both vertically and transversely to a thickness of 5 um during the preparation. Sections were stained with H&E as well as Masson's Trichromatic stain. All photomicrography taken was of 100 × magnification, scale bar 50 µm. Statistical analysis. The study used Graph Pad Prism software (version 6.04) to analyze the results. The Kolmogorov-Smirnov and Bartlett's tests were used to examine the data's normality and homogeneity of variance. The results are shown as the SD of the average of three replicates. To calculate the statistical significance www.nature.com/scientificreports/ between various groups, the study used one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Kruskal-Wallis non-parametric test, followed by multiple comparison Dunn's test was only used to determine the statistical significance of the hair follicle count histological score. The P value of ≤ 0.05 is significant throughout the manuscript.

Molecular docking study.
Preparation of protein receptor. The crystal structure of the androgen receptor in complex with minoxidil (PDB code:4K7A) was downloaded from http/www/pdbbeta.rscb.org with a resolution of 2.44 Ǻ. As DHT is a natural ligand that causes baldness and minoxidil is a drug used for the treatment of baldness, we used the minoxidil position in the crystal structure of the androgen receptor as the position for validation throughout the docking analysis. The receptor was 3D protonated, where hydrogen atoms were added at their standard geometry, the partial charges were computed, and the system was optimized. Deletion of cocrystallized water molecules was performed. The binding pocket had been defined and isolated (Fig. 2).
Validation of the molecular docking method. To ensure the accuracy of the docking protocol, validation of the molecular docking method in this study was performed by re-docking minoxidil in the active site of the target protein using AutodockVina and visualized by Discovery Studio Visualizer. This was followed by the alignment of the X-ray bioactive conformer of the minoxidil with the best fitted pose achieved from docking. The alignment showed good coincidence between them, indicating the ability of the used docking protocol to retrieve valid docking poses. The method was deemed successful if the RMSD value returned was ≤ 2 Å 15 (Fig. 3).
Preparation of ligand structure. Test ligand structures of LSLE compounds, minoxidil and the reference ligand finasteride were prepared using AutoDock Vina. The ligand setup was employed to add gasteriger charges, find aromatic/aliphatic carbons, detect rotatable bond, and set torsion angle. Finally, the compounds were saved as PDBQT format.
Running docking protocol. The docking analyses of the identified molecules with androgenic receptor (4K7A) were carried out using AutoDock Vina. The program was run using a searching grid. The grid was typically over the androgenic receptor and had a size of 20 Å × 20 Å × 20 Å. The other parameters were set as default. The center of the active site was determined and presented as a 3D grid, with X = − 24, Y = 1 and Z = − 3, respectively.   www.nature.com/scientificreports/ Compounds were docked into the crystal structure of 4K7A, and the highest scoring pose was selected for each of the compounds. The most stable conformation of each compound for binding to the protein active site was taken as the optimal docking posture.

LC-MS/MS of LSLE.
In folk medicine, heat is usually implemented in the extraction of L. sativum seeds.
Accordingly, there is a higher probability for the loss of active constituents, particularly glucosinolates 16 . These are ubiquitous metabolites of the Brassicaceae plants known for their hair growth promoting potential via the degradation of dihydrotestosterone as in case of sulforaphane 17 . Therefore, the use of heat was avoided, and seeds were soaked in cold water, then lyophilized to form LSLE. LC-MS/MS is an effective tool for the identification and dereplication of plant secondary metabolites 9,18 . Since plant extracts are constituting a complex matrix of diverse secondary metabolites with different chemical scaffolds that render their ionization behaviour different according to structure, we used both positive and negative ionization modes. Several glucosinolates has been reported and identified by LC-MS from plant extracts 19,20 . Data analysis resulted in the identification of 17 main compounds. Three glucosinolates, namely, glucobrassicanapin (14), glucotropeolin (5), sinigrin (3), seven flavonoids, namely, acacetin-7-O-rutinoside (16), catechin (10), (15), a phenolic acid, sinapic acid (2), together with its glucoside (12), semilepidinosides A and B (4 and 7), the alkaloid lepidiene E (6), the coumarin esculin (1), and linolenic acid (17) were detected (Table 1 and Fig. S1). LC-MS/MS analysis followed by molecular networking of LSLE helped leveraging the metabolites of L. sativum seeds. Molecular networks (MNs) for the negative and positive ionization modes were created via GNPS platform (Global Natural Products Social Molecular Networking), where MN displayed the chemical space acquired from MS-MS fragmentation patterns similarity to enable the correlation of probably similar metabolites. This correlation would accelerate sorting and dereplication of constituents of the metabolome 21 . The negative MN resulted in 114 nodes grouped as 7 clusters (Fig. 4). They were dereplicated as a set of Clusters were cluster A (flavonoids), cluster B (sugars), cluster C (phenolic acid glycosides), cluster D (myoinositol derivatives), cluster E (fatty acid), cluster F (phenolic acid esters) and cluster G (glucosinolates). On the other hand, positive MN revealed similar results (Fig. 5). glucosinolates were the major compounds identified in the extract, followed by flavonoid glycosides, phenolic acids derivatives and alkaloids.
The major compounds identified from LSLE and their corresponding fragmentation patterns are illustrated in Fig. 6 for negative ion mode and Fig. 7 for positive ion mode.

Biochemical parameters. Effect of LSLE on cholesterol levels in rats with induced alopecia; treatment and
withdrawal. The administration of Sustanon showed an approximately 20% decrease in the level of cholesterol compared to the control group. Treatment with LSLE did not affect the cholesterol level while minoxidil significantly decreased the cholesterol level. After stopping the treatment, the level of cholesterol increased in both groups. The level of cholesterol was insignificantly higher in the LSLE than in the minoxidil group (Fig. 8). In groups V and VI where the treatment was stopped, the levels of 5AR fell more in the LSLE compared to the minoxidil but remained significantly higher (Fig. 9).
Effect of LSLE on CTGF in rats with induced alopecia; treatment and withdrawal. Administration of Sustanon for 21 days significantly reduced the CTGF levels in group II compared to the control group by 2.64 folds. The topical coadministration of minoxidil with the S.C of Sustanon increased CTGF by 1.68 folds compared to group II. Treatment with LSLE increased the CTGF to exceed that of the control group to reach 501.47 ± 60.24 pg/ml. After stopping the treatment, the level of CTGF decreased in both groups V and VI but the decrease was 48.5% in the LSLE group compared to only 28% in the minoxidil group. The concentration remained relatively higher in the LSLE group (Fig. 10).   www.nature.com/scientificreports/ Effect of LSLE on FGF in rats with induced alopecia; treatment and withdrawal. FGF levels were measured by relative quantification against the housekeeping gene (β-actin) per tissue sample and relative to the control group. The group of induced alopecia manifested a significant reduction in the FGF levels by 76% compared to the control group. Nevertheless, both treated groups showed higher FGF levels. Group III was 13% higher while group IV was 40% higher compared to group II. On discontinuing the treatment, the levels of FGF were normalized to the minoxidil group (group V). The level of FGF was 75% higher in group VI compared to group V (Fig. 11).
Effect of LSLE on VEGF in rats with induced alopecia; treatment and withdrawal. The administration of Sustanon decreased the VEGF expression levels by 5 folds compared to the control group. On treating the rats, the VEGF levels increased significantly by 2.5 folds in the minoxidil group and by 5 folds in the LSLE group com-   www.nature.com/scientificreports/ pared to the untreated group. After stopping the treatment, the level of VEGF decreased in both groups, V and VI. The level in the minoxidil group decreased by 20% and in the LSLE by 25% (Fig. 12).
Hair growth and histopathology. Hair growth. Hair growth rates were observed daily and by comparing the hair growth among the groups. The LSLE showed the highest hair growth rate (Fig. 13).
Histopathological assessment of hair follicles. Transverse sections are a very promising and reliable approach for confirming the diagnosis because all hair follicles can be clearly visualized, so all hair follicles were counted on transverse sections for greater accuracy. The total number of hair follicles may be unaffected, but the effect was observed as progressive miniaturization in the already present hair in the control positive group, as well as a difference in the size of hair follicles and expanded vellus hairs (growing hair-thin yellow arrows) in the tested groups. The total number of hair follicles counted along 10 examined fields in each group was statistically calculated (Figs. 14 and 15). The findings were confirmed on Masson trichrome stained sections, which allowed for a better view of the growing hair follicles. The 5AR levels decreased slightly to 1.24 ± 0.364 ng/ ml in group V compared to 1.8 ± 0.356 ng/ml in group VI. Statistical analysis was done using one-way ANOVA followed by Tukey's post hoc test. # refers to a significant difference from the untreated group (p < 0.05). * Refers to a significant difference from the minoxidil treatment group (p < 0.05).  www.nature.com/scientificreports/ On continuation of the study for the further effect of both drugs, H&E-stained sections of treated groups revealed excess hair follicular growth as well as long hair shafts with a privilege of LSLE over minoxidil treated groups. Representative Masson trichrome microscopic images confirmed the ordinary H&E results with better visualization of dermal papillae, hair shafts and bulbs (Figs. 16 and 17).
Docking study. Docking simulation of the major ten compounds identified showed that they fit into the receptor active site almost at the same position of minoxidil with comparable docking scores ranging (from − 4.1 to − 8.9 kcal/mol, in comparison with − 6.9 kcal/mol for minoxidil) (Fig. 18, Table 2).
Minoxidil and the androgen receptor form hydrogen bonds at GLU 793 , with LEU 862 , LYS 861 , and TYR 857 being the four nearest amino acid residues. Different amino acid residues creating a hydrogen bond with the androgen receptor are factors that cause the binding energy for finasteride and identified compounds to be higher than minoxidil. Minoxidil forms hydrogen bonds with the androgen receptor's GLU 793 . Additional hydrophobic interactions with TRP 796 and HIS 789 influence ligand stability with the androgen receptor. Hydrophobic interactions, which repel liquid, are more likely to gather in protein globular form. Arginine is projected to play a crucial function in the androgen receptor ligand binding domain 34   The results were normalized relative to the minoxidil group. The concentration was relatively higher in the LSLE group than in the minoxidil group. Results represent mean ± SD (n = 7). Statistical analysis was done using one-way ANOVA followed by Tukey's post hoc test. # Refers to a significant difference from the untreated group (p < 0.05). * Refers to a significant difference from the minoxidil treatment group (p < 0.05). (A) Effect of treatment. Group I had the highest level of tissue VEGF with a ratio of 1.12 ± 0.08 to β-actin. Administration of Sustanon resulted in decreasing the VEGF level to 0.21 ± 0.05. Treatment with minoxidil and LSLE increased the VEGF levels to 0.53 ± 0.1 and 1.08 ± 0.12 respectively. (B) Effect of stopping the treatment. The level of VEGF fell back to 0.42 ± 0.1 in group V and 0.76 ± 0.09. The concentration was relatively higher in the LSLE group than in the minoxidil group. Results represent mean ± SD (n = 7). Statistical analysis was done using one-way ANOVA followed by Tukey's post hoc test. # Refers to a significant difference from the untreated group (p < 0.05). * Refers to a significant difference from the minoxidil treatment group (p < 0.05).  damaged hair follicles no longer able to grow to a developed hair shaft) in control positive group "yellow arrows" (C) Lepidium tested group with high number of active growing follicles resembling normal group "yellow arrows" (D) Minoxidil tested group visualizing multiple empty follicles than found in Lepidium group but less than control positive group "yellow arrows".

Discussion
Androgenic alopecia (AGA) is a difficult condition that affects both men and women. It is tolerated by some, but it lowers their self-esteem and has a negative psychological impact on them. This study was simply designed to investigate the therapeutic effect of LSLE on AGA and compare it to minoxidil 5%, which is available on the market and used to treat similar cases. A modification in the extraction of the L. sativum seeds' active constituents was performed. The seeds were soaked in cold water and then lyophilized to form LSLE. The traditional use of heat treatment in folk medicine was avoided to prevent the loss of active constituents.
Glucosinolates are repeatedly reported to alleviate AGA conditions by enhancing the degradation of DHT 17 , inhibiting testosterone induced dermal papillae cells (DPCs) apoptosis 5 , stimulating DPCs proliferation and upregulating vascular endothelial growth factor (VEGF) 6 , or antiandrogenic activity 8 . Which postulates that the LSLE glucosinolates have a crucial role in promoting hair growth.
Meanwhile, flavonoids and phenolic acids are well known antioxidants that can alleviate oxidative stress. Oxidative stress in turn can cause many adverse effects on the skin and scalp that accelerate hair loss 35 .
On the other hand, docking study of ten representative constituents of LSLE against the androgen receptor (PDB code: 4K7A). revealed highest binding energy (ΔG) for the flavonoids luteolin-3' ,7-dihexoside, quercetin 3-O-deoxyhexose-hexose-7-O-deoxyhexose, the alkaloidal glycosides semilepidinoside B and semilepidinoside A when compared to the standard drug minoxidil, which is mostly attributed to hydrogen bonding with amino Figure 15. Photomicrography with Masson trichrome stain transverse sections of rat's skin: (A) Normal control group showing the active hair follicles "yellow arrows" with deeply stained collagen around in blue green color, (B) Resting dorminant follicles in control positive group that are empty with no further hair follicles "yellow arrows" (C) LSLE tested group with higher presence of active growing follicles resembling normal tested groups "yellow arrows" (D) Minoxidil tested group visualizing multiple empty follicles exceeding that found in the LSLE group but much less than control positive group "yellow arrows". www.nature.com/scientificreports/ acids that exist in vicinity of the ligand site, in addition to GLU 793 , which is the seldom hydrogen bonding residue with minoxidil. For the aforementioned reasons regarding the diversity of metabolites in LSLE and its correlation to their antiandrogenic, antioxidant and predicted AR inhibitory activity, the use of the LSLE might be useful for the treatment of AGA without need for further fractionation or purification, due to the reciprocating potential of its diverse metabolome to aim multiple targets involved in the etiology of AGA.
Sustanon is made up of four different testosterone esters (testosterone propionate, phenylpropionate, isocaproate, and decanoate) 36 . These are simply short and intermediate esters with shorter absorption half-lives and higher clearance rates than longer chain esters 36 . As a result, Sustanon (a testosterone analogue) was used in our study to induce alopecia quickly.
The level of cholesterol was inversely proportional to the level of testosterone. This was reported by Zarei et al. 37 , and can be attributed to the conversion of the testosterone to 17-β-estradiol 38 . Although this remains controversial as other studies stated that testosterone may activate HMG-CoA reductase 39 , we were interested more in the fact that minoxidil reduced serum cholesterol. It was reported in a previous study that systemic minoxidil can affect cholesterol levels 40 and so, it seems that there is partial absorption from its topical application. This was discussed before where around 1.7% of the applied minoxidil is systemically absorbed 41 . Which explains why the cholesterol levels were lower in groups III and V compared to groups IV and VI. This is advantageous to the LSLE as it showed no detected systemic effect.
5AR, which converts testosterone to DHT, is an important enzyme in determining the rate of androgenic alopecia. DHT has been shown to increase the expression of 5AR 42 , which explains the elevated level in group II. Minoxidil reduced 5AR concentrations, but the reduction was minor and insignificant when compared to the untreated group. This minor reduction can be attributed to the fact that minoxidil has been shown to inhibit 5AR expression in keratinocyte cell lines 43 . LSLE was able to decrease the concentration of 5AR in the skin homogenate which could account for the rapid hair growth observed in the mice after 21 days and may be the reason why members from the same family of LSLE have been used for centuries as hair tonics 44 . After stopping the treatment, the LSLE remained for 4 weeks as the concentration of 5AR was still below that of group II despite the administration of Sustanon.
AGA has commonly been characterized by a marked decline in the supply of blood, oxygen, and nutrients to the hair follicles. It is worth mentioning that DHT binds to androgen receptors on the hair follicles causing apoptosis of the dermal papilla (DP cells), disrupting the proliferation of keratinocytes and impairing the action of growth factors 45 . We herein evaluated the levels of three fundamental growth factors (VEGF-CTGF-FGF) for hair www.nature.com/scientificreports/ growth amongst different treatment groups. These factors play a key role in promoting vascularization, fostering angiogenesis, and thereby enriching the follicles with the nutrients and oxygen required for hair growth [46][47][48] . Vascular endothelial growth factor (VEGF) is an autocrine growth factor that acts by directly interacting with hair DP cells 49,50 , resulting in increased follicle diameter 51 . It is a biological marker for hair follicle stimulation and growth 51 . Minoxidil, an FDA-approved alopecia treatment, has been recognized for its ability to increase the expression of VEGF in hair DP cells, which stimulates hair growth 52 . Nonetheless, the therapeutic effects of topical Minoxidil 5% can be reversed or reduced following treatment discontinuation 53 .
In the current study, the LSLE-Sustanon group demonstrated the highest levels of VEGF (5-folds). This increase was significantly higher than that observed with Minoxidil 5%-Sustanon group (2.5-folds) and remained so even after treatment discontinuation. As such, our findings suggest that LSLE possess a robust potential for the treatment of androgenic alopecia and produces a more prolonged therapeutic effect than Minoxidil.
The connective tissue growth factor (CTGF, or CCN2) is a matricellular protein that actuates other Extracellular Matrix (ECM)-proteins such as VEGF, transforming growth factor-β (TGF-β), integrin receptors, fibronectin, type-I collagen, and mucins [54][55][56] . Accordingly, CTGF serves multiple biological functions such as angiogenesis, cell growth and tissue repair. Animal studies established a strong association between deficiency in the CTGF gene product and development of alopecia in transgenic mice 57 . The knockout CTGF mice also demonstrated deterioration in the angiogenesis process 56 .
In this study, Sustanon-only group (where alopecia was induced without any treatments) demonstrated the lowest CTGF level which lies in accordance with previous studies. Meanwhile, the CTGF level of the LSLE-Sustanon group surpassed that of Minoxidil 5%-Sustanon group even after discontinuation of treatment. The exceedingly high level of CTGF observed following LSLE treatment, as well as the persistence of such an elevated CTGF level after treatment cessation-further asserts its effectiveness for use against androgenic alopecia.
The connective tissue growth factor (FGF) usually exists among other growth factors like TGF-β, VEGF, and epidermal growth factor in platelets of blood plasma. The release of these growth factors is crucial to bolster the differentiation of stem cells into hair follicles by upregulating the transcription of β-catenin 58 . These factors also www.nature.com/scientificreports/ possess an anti-apoptotic activity by stimulating the anti-apoptotic signaling pathways including Bcl-2 and Akt, hence, interrupting the catagen phase while prolonging the anagen phase and promoting hair growth 45 . Our study showed that FGF levels of the LSLE-Sustanon group exceeded that of Minoxidil 5%-Sustanon group even after treatment discontinuation which again reinforces the beneficial therapeutic effects of LSLE in androgenic alopecia. Figure 18. Visualization of interactions between the androgen receptor (4K7A) and ten major compounds identified showed comparable or better binding energy and binding modes than that of minoxidil (6.9 kcal/mol) as well as that of the gold standard therapy compound, finasteride.  59 . This can be attributed to a variety of reasons; the relatively high cost of the drug 60 , the adverse effects that follow its application such as pruritus, rash, dandruff, desquamation, allergic contact dermatitis 60,61 and systemic hypotension 62 , in addition to the long time it takes for the results to be apparent whereby the patient may wait for up to 6 months to obtain a noticeable effect 53 . In a number of reputable papers that were published, it was stated that topical application of 5% minoxidil might have caused non-arteritic anterior ischemic optic neuropathy that was cured after the treatment stopped 63 . Consequently, it is important to search for better alternatives.
It is noteworthy to mention that compared to the approved drug Minoxidil, LSLE extract possessed higher inhibitory activity on 5α-reductase and resulted in significantly higher levels of the examined growth factors (VEGF, CTGF and FGF) even after treatment cessation. The immense increase in growth factors following LSLE application secures the supply of oxygen, blood and nutrients to the scalp and lowers the rate of alopecia.
In conclusion, the present study gives an insight about the promising hair-growth and anti-androgenic effects of LSLE. Further research on the anti-apoptotic activity, as well as the systemic absorption of LSLE may be needed to unravel the underlying molecular mechanisms and ensure its safety.

Data availability
The datasets analyzed during the current study will be available from the corresponding author on reasonable request.